The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred′H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the “dithionite (DT) inhibited” states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.


■ INTRODUCTION
[FeFe] hydrogenases are highly active metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen. 1,2 Their active site, the H-cluster, comprises a unique diiron subcluster ([2Fe] H ) and a canonical [4Fe-4S] subcluster ([4Fe-4S] H ), covalently linked by a cysteine thiolate 3,4 (Figure 1 A and B). The Fe of [2Fe] H that is closest to [4Fe-4S] H is known as the proximal Fe (Fe p ), while the Fe furthest from the cluster is known as the distal Fe (Fe d ). In [2Fe] H , the Fe ions are coordinated by two terminal CN − and two terminal CO ligands (one on each Fe), a bridging CO, and a bridging 2-azapropane-1,3-dithiolate (ADT) ligand. 5,6 During H 2 conversion, the H-cluster goes through a series of redox transitions, where the Fe ions change oxidation states, as well as protonation/deprotonation steps. 7−9 While several catalytic intermediate states have been well characterized with a variety of spectroscopic techniques, structural models based on X-ray diffraction data on crystals in spectroscopically defined states are not generally available. Thus, in the absence of structural models supported by experimental data, computational chemistry has played an important role in proposing likely structures of the active site in the catalytic intermediates based on spectroscopic data. However, divergent results from various groups have led to several possible models of the catalytic cycle of [FeFe] hydrogenases. 8,10−12 These can be summarized in two main models (here referred to as Model 1 and 2, Figure 1C and D respectively).
The most oxidized state of the active enzyme, H ox , is generally accepted to be the starting point of the catalytic cycle and has a mixed valence of Fe p (II)Fe d (I) in [2Fe] H , 13 and an oxidized [4Fe-4S] H 2+ . In Model 1 ( Figure 1C), one-electron reduction of H ox is proposed to yield two possible states H red and H red H + (in our nomenclature), whose relative population depends on the pH. In H red , the electron is thought to be localized preferentially on [4Fe-4S] H . In H red H + , the electron is thought to be transferred to the [2Fe] H subcluster (with an Fe p (I)Fe d (I) configuration) and a proton (from the proton transfer pathway) to bind to the nitrogen in the ADT bridge giving an NH 2 + . 14,15 This process of proton-coupled electronic rearrangement (PCER) of the H-cluster is a crucial component of Model 1. A further one-electron reduction of H red H + yields the H sred H + state with a reduced [4Fe-4S] H + . 16,17 The protonated ADT ligand in both H red H + and H sred H + appears to be able to transfer the proton to Fe d generating an Fe-bound hydride in the H hyd state. 18−23 Finally, the H hyd state is thought to gain an additional proton, which may trigger a similar PCER process as in the H red state, and form a H 2 molecule bound to Fe d , which can then leave the enzyme via a hydrophobic gas channel. 24 Recently, photoexcitation of H red H + and H sred H + was shown to generate two different forms of H hyd , known as H hyd:ox and H hyd:red , where the former has an oxidized [4Fe-4S] H and the latter has a reduced [4Fe-4S] H . 25 In Model 2 ( Figure 1D), 8 the H red state (referred to as H red ′) is formed from H ox by proton-coupled electron transfer (PCET) at [4Fe-4S] H . This was suggested based on IR spectroelectrochemical titrations at various pH values that showed a pH-dependent redox potential of [4Fe-4S] H . 10 Specifically, the proton is thought to bind to one of the cysteine ligands coordinating the cluster. This state then undergoes an additional PCET at [2Fe] H to give the H hyd state, 19 and the proton is retained on [4Fe-4S] H . Hydrogen is then formed by additional protonation leaving an oxidized H-cluster but still protonated at [4Fe-4S] H , a state called H ox H. 27 In Model 2, H red H + (referred to as H red ) and H sred H + (not shown in Figure 1D) contain a bridging hydride (μH − ) and an apical CO ligand, 28 and are considered to be part of a low activity pathway. Lastly, reduction of H ox H to H red ′H has also been observed, 10 but its place in the catalytic cycle remains to be determined.
The H ox H and H red ′H states in Model 2 have been reported to accumulate at low pH only in the presence of sodium dithionite (NaDT) (see Supporting Information for further details). 10,27 NaDT (Na 2 S 2 O 4 , also sodium hydrosulfite) is widely used in biochemistry as an oxygen scavenger and low potential reducing agent (E 0 ' = −0.66 V vs SHE at pH 7 and 25°C). 29 For example, it is commonly employed to protect metalloproteins from oxidative damage caused by trace amounts of oxygen during purification and handling, or to poise metallocofactors in reduced states for their characterization. However, one of the pitfalls of its use is the failure to consider that NaDT and its oxidation products can engage in side-reactions with the system under study. Several studies on sulfite-reducing enzymes have highlighted how oxidation of NaDT can be a significant source of SO 3 2− , the substrate for these enzymes, which can bind to the active site and   27 Alternatively, H red ′ can rearrange to give a less active H red state containing a bridging hydride, 28 which proceeds through a low activity pathway. H ox H appears to undergo one-electron reduction to H red ′H, but this is not included in the catalytic cycle. 10,27 complicate the interpretation of spectroscopic studies and activity measurements. 30−32 In a recent report, during the semisynthetic assembly of the FeMo cofactor of nitrogenase, the donor of the ninth sulfur ligand was found to be the SO 3 2− generated by the oxidation or degradation of NaDT present in the assay. 33 Numerous studies have reported the interaction of oxidation products of NaDT with various enzymes including nitrite reductase, 34−36 DMSO reductase, 37 monomethylamine methyltransferase, 38 acetyl CoA synthase, 39 and formation of adducts to flavins 40−42 and cobalamin. 43,44 Additionally, the slow dissociation of NaDT into SO 2 •− radicals (the active reducing species) has been shown to be problematic in mechanistic studies of nitrogenase. 45 In light of the dependence of H ox H and H red ′H on NaDT, and of NaDT's reported "non-innocent" behavior, we decided to investigate the effect of NaDT and its oxidation products on [FeFe]  hydrogenases. Furthermore, these findings highlight the importance of carefully considering the possible side-reactions of NaDT and its oxidation products when choosing to use this reducing agent with metalloenzymes, particularly iron−sulfur enzymes.

■ RESULTS
Treatment of CrHydA1 with oxidized NaDT causes formation of the HoxH state. Our investigation on the effect of the oxidation products of NaDT on [FeFe] hydrogenases focused, in the first instance, on CrHydA1, the most well characterized [FeFe] hydrogenase, which contains only the H-cluster. In particular, the enzyme containing the native [2Fe] cofactor with the ADT ligand (CrHydA1 ADT ) was used. Thus, CrHydA1 ADT produced in the strict absence of NaDT was treated with a solution of oxidized NaDT (oxNaDT). This solution was prepared by dissolving fresh NaDT in water to a concentration of 1 M (the most effective concentration of NaDT for H ox H formation at pH 6 27 ) under aerobic conditions and stirring for 2 h under atmospheric oxygen. A decrease in the pH to ∼2 and appearance of a yellow precipitate (most likely elemental sulfur) indicated oxidation and degradation of the dithionite anion. 47 The oxNaDT solution was then thoroughly degassed and moved into an anaerobic glovebox before being added to CrHydA1 ADT , in order to avoid damaging the highly air-sensitive H-cluster.
As shown by the IR spectra of CrHydA1 ADT (Figure 2), dilution of the enzyme in the oxNaDT solution results in the appearance of a new set of vibrational signals, slightly shifted to higher energy (<10 cm −1 ) with respect to H ox . These new signals are consistent with those reported for H ox H. 10,19,27 Even though the pH of the oxNaDT solution was measured to be around 2, the buffer present in the CrHydA1 ADT sample (25 mM Tris-HCl, pH 8) will render the pH value after oxNaDT addition slightly higher than this (ca. pH 6). Interestingly, when CrHydA1 ADT was treated with a solution oxNaDT whose pH had been corrected to 7, conversion to H ox H was not observed ( Figure S1), suggesting that formation of this state requires acidic conditions.
The observation that H ox H can be formed by treatment with oxidized NaDT challenges the hypothesis that H ox H and H red ′H are protonated versions of the H-cluster. However, it was not clear which component of oxNaDT was interacting with the H-cluster. To better understand the nature of these two states and their role in the catalytic cycle of [FeFe] hydrogenases, we sought to identify the oxidation product(s) of NaDT responsible for their formation.
H ox H forms in the presence of sulfite at low pH. The main oxidation and degradation products of NaDT are sulfate Spectra were normalized to allow easier comparison from different measurements. Peaks for the H ox and H ox H states are highlighted in blue and red, respectively. Small contributions from the H ox -CO (gray asterisk), H red H + (green), and H red (black) states are indicated. Importantly, Na 2 SO 3 solutions were pH corrected before use, whereas the solution of oxNaDT was not pH corrected, but measured to be around 2.
(SO 4 2− ), thiosulfate (S 2 O 3 2− ), and sulfite (SO 3 2− ), 48,49 all of which could potentially interact with the H-cluster of CrHydA1 and cause conversion to the H ox H state. Therefore, to identify the NaDT oxidation products responsible for this conversion, we tested these species individually on CrHydA1 at both pH 8 and pH 5. Treatment of CrHydA1 with Na 2 SO 4 and Na 2 S 2 O 3 at either pH 8 or pH 5 failed to reproduce the H ox H state ( Figure S2). In contrast, we found that addition of 80 mM of Na 2 SO 3 at pH 5 reproduced the effect of oxNaDT and caused almost full conversion to the H ox H state, while at pH 8 even a high concentration (0.92 M) of Na 2 SO 3 had no effect on CrHydA1 ( Figure 2). Importantly, CrHydA1 at pH 5 before addition of Na 2 SO 3 has an identical spectrum to that at pH 8, demonstrating that both low pH and Na 2 SO 3 are required for H ox H formation. Na 2 SO 4 , Na 2 S 2 O 3 , and Na 2 SO 3 solutions were pH corrected before usethis is particularly important for Na 2 SO 3 , which is a mild base.
In addition to CrHydA1, also the bacterial [FeFe] hydrogenases HydAB from Desulfovibrio desulf uricans (DdHydAB) and HydA1 from Clostridium pasteurianum (CpHydA1) have been reported to form the H ox H state at low pH and in the presence of NaDT. 27 These enzymes harbor additional [4Fe-4S] clusters (F-clusters) that form an electrontransfer chain from the protein surface to the H-cluster, and compared to CrHydA1, their active site is deeply buried inside the protein scaffold. 3,4 When treated with Na 2 SO 3 under acidic conditions, also DdHydAB and CpHydA1 converted to the H ox H state ( Figure S3), indicating that the interaction of the H-cluster with the oxidation product of NaDT is a generalized phenomenon in [FeFe] hydrogenases.
A protonated form of sulfite interacts with the H-cluster. Next, we decided to carry out titrations of CrHydA1 with Na 2 SO 3 at various pH values in order to provide further details on the particular form of Na 2 SO 3 that binds, as well as determining the binding affinity. In order to simplify the titrations, we chose to use a chemical variant of CrHydA1 with a [2Fe] H analogue containing a propane dithiolate (PDT) bridging ligand instead of ADT (CrHy-dA1 PDT , Figure 1A). Compared to the amine in ADT, the methylene group in PDT cannot be easily protonated. As a result, CrHydA1 PDT has very low catalytic activity and the Hcluster cannot assume states with a reduced [2Fe] H (i.e., H red H + and H sred H + ) ( Figure 1C). This greatly reduces the number of states observable in the IR spectra, simplifying data analysis. The PDT-containing enzyme was previously shown to convert to H ox H and H red ′H at low pH in the presence of NaDT. 10,27 CrHydA1 PDT was titrated with increasing amounts of Na 2 SO 3 at five different pH values ( Figure 3 and Figures  S4−S6). In an anaerobic glovebox with a 100% N 2 atmosphere, the H-cluster was in the oxidized state H ox at the beginning of the titration for all the pH values tested. As already observed for native CrHydA1 ADT , at pH 8 addition of even a very high concentration of Na 2 SO 3 did not affect the state of the Hcluster, which remained in the H ox state. Conversely, at pH 7, H ox H appeared already with less than 250 mM Na 2 SO 3 , and complete conversion was observed at around 700 mM. The concentration of Na 2 SO 3 needed in order to observe complete Predicted speciation of sulfite in water as a function of the pH assuming an acid dissociation constant (pK a ) of 7.19 for HSO 3 − ⇌ H + + SO 3 2− and an equilibrium constant (pK) of 1.76 for SO 2 + H 2 O ⇌ H + + HSO 3 − . 50 (C) Variation in the intensity of the 1942 cm −1 (H ox ) and 1946 cm −1 (H ox H) peaks with the estimated concentration of dissolved SO 2 at pH 7 (triangles) and 6 (circles). The data were fitted with a model describing binding of SO 2 to the hydrogenase with 1:1 stoichiometry and assuming that the concentration of SO 2 at equilibrium is determined only by the pH and the concentration of Na 2 SO 3 . The data at pH 6 and 7 were fitted simultaneously to the same model. For an expanded version of the region from 0 to 6 μM SO 2 ; see Figure S6E. Error bars (±standard deviation) were determined by measuring the 0, 0.25, and 0.92 M Na 2 SO 3 spectra at pH 7 and the 25 mM Na 2 SO 3 spectrum at pH 6 in triplicate, which gave standard deviations of less than 0.014.
conversion from H ox to H ox H decreased at pH 6 to about 200 mM and at pH 5 to less than 8 mM. At pH 4, 1 mM Na 2 SO 3 gave essentially complete conversion to H ox H, while 1 mM Na 2 SO 3 at pH 5 gave a roughly equal mixture of H ox and H ox H ( Figure S5).
In aqueous solutions SO 3 2− is in equilibrium with its protonated form bisulfite (HSO 3 − ), which in turn can be further protonated to form sulfurous acid (H 2 SO 3 ), which immediately decomposes to sulfur dioxide (SO 2 ) and water ( Figure 3B). 51−53 As Figure 3 shows, the lower the pH, the lower the concentration of sulfite needed to convert H ox to H ox H. This, therefore, excludes that SO 3 2− , whose abundance is predicted to greatly decrease when changing the pH from 8 to 6, is responsible for formation of H ox H. Since, as shown in Figure 3A, lowering the pH from 6 to 5, and then to 4 ( Figure  S5), caused a further reduction in the required concentration of Na 2 SO 3 needed to convert H ox to H ox H, while the fraction of HSO 3 − should be constant in this range ( Figure 3B), HSO 3 − is also unlikely to be the form of Na 2 SO 3 binding to the H-cluster. In a pH titration of Na 2 SO 3 monitored by IR spectroscopy we observed that the intensity of peaks relative to HSO 3 − indeed saturated after pH 6.0−5.5, while signals indicative of the presence of SO 2 appeared at pH 5 ( Figure  S7). Therefore, we hypothesize that the species interacting with the H-cluster to form H ox H is SO 2 . This seems reasonable considering that SO 2 is a neutral molecule able to easily diffuse through hydrophobic channels 54,55 to reach the H-cluster from the protein surface, while the anions HSO 3 − and SO 3 2− will be prevented from entering due to their charge and their large hydration spheres in aqueous solution. 56 A similar suggestion was made to explain how S 2− reaches the H-cluster as H 2 S to form the H inact state. 57 At pH 7 and 6, even at high concentration of sulfite, the concentration of dissolved SO 2 is expected to be very low. Thus, in order to observe binding to the H-cluster and formation of H ox H, SO 2 must have a tight affinity for the enzyme. Figure 3C shows the conversion from H ox to H ox H as a function of the estimated concentration of SO 2 at each Na 2 SO 3 addition, at either pH 6 or 7. The population of the two states was monitored from the intensity of the most prominent CO band at 1942 cm −1 for H ox and 1946 cm −1 for H ox H, in both cases corresponding to the stretch of the terminal CO on Fe d . The titration curves at pH 7 and 6 as a function of the concentration of SO 2 overlay nicely, in contrast to those obtained using the estimated concentrations of HSO 3 − and SO 3 2− ( Figure S6). Fitting the data in Figure 3C to a simple equilibrium model describing one SO 2 molecule binding to the hydrogenase (SO 2 + E ⇌ E:SO 2 ) gave an estimated binding affinity of ∼500 nM. In our analysis, we considered that the pool of Na 2 SO 3 can act as a buffer system for SO 2 , replenishing what is consumed to form the enzyme:SO 2 complex (E:SO 2 ). For all the data points, the concentration of E:SO 2 formed was negligible compared to the total concentration of Na 2 SO 3 , so that the concentration of SO 2 at equilibrium could be assumed to be independent of the formation of E:SO 2 and to be determined only by the pH and the total concentration of Na 2 SO 3 , an important consideration for such tight binding interactions. To put this in context, CO has been estimated to bind with 100 nM affinity to CrHydA1 ADT . 58 Addition of sulfite under reducing conditions (H 2 atmosphere) forms H red ′H. The titration of CrHydA1 PDT with sulfite was repeated in the presence of 2% H 2 in the atmosphere of the anaerobic glovebox (Figure 4). Under these conditions, slow reactivity of the CrHydA1 PDT enzyme with H 2 can lead to reduction of the [4Fe-4S] H subcluster, in particular at high pH values. This is due to the potential of the 2H + /H 2 couple, which becomes more positive as the pH decreases, while the redox potential of [4Fe-4S] H is pH independent. 12 At pH 7, after addition of a small amount of Na 2 SO 3 , we observed a mixture of the H ox , H red , and H ox H states in the IR spectra, plus a new set of signals. These are consistent with the vibrational frequencies of the H red ′H state, which Stripp and co-workers reported to form with NaDT at low pH and either under H 2 or at low electrochemical potential. 10,27 Similar to what was observed under N 2 , at lower pH the formation of H ox H and H red ′H was observed at lower concentration of Na 2 SO 3 . In order to estimate the proportion of each state present under each condition, we performed a pseudo-Voigt peak-fitting analysis of the region of the spectrum between ∼1955 cm −1 and ∼1920 cm −1 , containing the most dominant bands for H ox (1942 cm −1 , blue), H red (1935 cm −1 , cyan), H ox H (1946 cm −1 , red), and H red ′H (1939 cm −1 , purple) ( Figures 4B, S8−S10). In Figure 4C, the intensity of these contributions is plotted as a function of the concentration of Na 2 SO 3 at pH 7. At low Na 2 SO 3 , both the H ox H and H red ′H states are observed, but at high concentrations of Na 2 SO 3 H red ′H is converted to H ox H. This indicates oxidation of the [4Fe-4S] H subcluster by Na 2 SO 3 . Since the samples were prepared in a closed IR cell and the concentrations of sulfite used are much higher than the dissolved concentration of H 2 , oxidation by Na 2 SO 3 will slowly deplete the H 2 concentration leading to oxidation of the sample. Similar behavior is observed also at pH 6 and 5 ( Figures S8, S10).
At low concentrations (8 mM) of Na 2 SO 3 , at pH 6, the H red ′H is the most dominant state, while H ox H becomes more favored at pH 5 at the same concentration of Na 2 SO 3 , agreeing with a pH independent redox potential of [4Fe-4S] H also when SO 2 is bound. However, the fact that SO 2 is more prevalent at low pH gives the conversion of H ox /H red to H ox H/H red ′H an "apparent" pH dependence. This will complicate the interpretation of pH-dependent redox titrations performed in the presence of oxidation products of sodium dithionite (including Na 2 SO 3 and SO 2 ), which may explain discrepancies in the literature. 10,12 Interestingly, at low concentrations of Na 2 SO 3 , the ratio of H ox :H red is much greater than that of H ox H:H red ′H, suggesting that binding of SO 2 increases the redox potential of the [4Fe-4S] H subcluster ( Figure 4B and S8, S9). The redox potential for the H ox /H red and H ox H/H red ′H transitions can be calculated at pH 6 and 7 at low concentrations of Na 2 SO 3 from the populations of the four states ( Figure S11). Using the Nernst equation, we found that E m (H ox /H red ) = −349 (±17) mV and E m (H ox H/H red ′H) = −293 (±26) mV. The value for E m (H ox /H red ) is in close agreement with that determined previously. 12,59 The fact that the redox potential for the H ox H/ H red ′H transition is ∼60 mV more positive than the H ox /H red transition also indicates a tighter binding affinity for SO 2 Figure 4B with Figure  S4B).  60 At pH 5 exposure of CrHydA1 ADT to 100% CO gas for 10 min in the absence of Na 2 SO 3 generates pure H ox -CO ( Figure S12). In the presence of a high concentration of sulfite at pH 5, exposure of CrHydA1 to CO caused the appearance of new peaks that correspond to neither H ox -CO nor H ox H, and are similar to the H ox H−CO state described by Stripp and co-workers ( Figure S12). 27 This suggests that SO 2 does not compete for the same binding site as CO, which is the open coordination site at Fe d .
In order to get further information on the SO 2 binding site, we measured 57 Fe nuclear resonance vibrational spectroscopy (NRVS). This technique measures Fe-ligand vibrational energies using nuclear excitation of 57 Fe and has been used extensively to probe ligand binding to the [2Fe] H subcluster in [FeFe] hydrogenase. [21][22][23]28,57 We artificially maturated apo-CrHydA1 samples with a 57 Fe-labeled diiron subcluster precursor ([2 57 Fe] ADT ) and measured NRVS in the H ox and H ox H states ( Figure 5). This enzyme is labeled with 57 [FeFe] hydrogenase. In order to investigate the effect of SO 2 -binding to the H-cluster on catalysis, we performed protein film electrochemistry on the DdHydAB enzyme covalently attached to a pyrolytic graphite electrode. We chose DdHydAB rather than CrHydA1, as the former is, in our hands, much easier to covalently attach to graphite electrode surfaces. 62 As shown in the cyclic voltammograms (CVs) in Figure 6 and in the enlarged version of the CVs reported in Figure S13, a large negative current at low potentials is observed when Na 2 SO 3 is injected into the electrochemical cell under acidic conditions (pH 5 and pH 6, respectively A and B in Figure 6). Controls experiments (bare graphite electrode injecting Na 2 SO 3 , Figure S14) suggest that this reduction current is likely due to HSO 3 − and SO 2 being reduced by the pyrolytic graphite electrode. 63 Comparisons of bare graphite electrodes and DdHydAB-modified electrodes at various pH values are presented in the absence ( Figure S15) and presence ( Figure S16) of Na 2 SO 3 . Unfortunately, this massive reduction current masks the effect of Na 2 SO 3 on the catalytic H + -reduction current.
However, as shown in Figure 6A and B (CV at pH 5 and 6 in the presence of Na 2 SO 3 , respectively), in the presence of Na 2 SO 3 the catalytic H 2 -oxidation current decreases, suggesting inhibition of the enzyme as a result of the H-cluster somehow interacting with SO 2 . The inhibitory effect on the catalytic H 2 -oxidation current is more pronounced at lower pH (the CVs at pH 7 and 8 are reported in Figure 6C and D, respectively), in agreement with the pH-dependent formation of H ox H and H red ′H observed in the IR measurements. To explore whether the inhibition is reversible and the electrocatalytic H 2 -oxidation current can be recovered, the buffer in   Figure S13. the electrochemical cell was exchanged to a fresh buffer without Na 2 SO 3 during the course of the CVs. Sulfite-exposed DdHydAB recovered 100% of the electrocatalytic H 2 -oxidation current once Na 2 SO 3 was removed from the electrochemical cell, suggesting that SO 2 binding and inhibition are fully reversible (blue trace in Figure 6A and B) and that the enzyme is not irreversibly damaged by SO 2 .
The massive current at low potential due to direct reduction of HSO 3 − and SO 2 species by the electrode makes it difficult to assess the effect of Na 2 SO 3 on the electrocatalytic H +reduction current. To distinguish the enzymatic contribution from the direct HSO 3 − and SO 2 reduction by the electrode, we performed chronoamperometry experiments (the applied potential is held at a specific value while the current is monitored vs time) in the presence and absence of CO ( Figure  7). As previously described, 58,62 the current decrease due to CO addition (as CO binds to open coordination site on Fe d and inhibits the enzyme) provides a direct measurement of the enzymatic H + reduction. In the experiment in Figure 7A, performed at pH 5, DdHydAB attached on the pyrolytic graphite electrode was initially exposed to 90% H 2 /10% N 2 at −109 mV vs SHE, where a positive current due to H 2 oxidation was observed (as the applied potential is more positive than the thermodynamic potential of the 2H +/ H 2 couple at this pH, −295 mV vs SHE). Switching to −459 mV gave a small negative current due to H + reduction (as the applied potential is now more negative than E 2H + /H 2 at this pH). Adding Na 2 SO 3 at this potential gave an extremely large negative current, which was unaffected by addition of 10% CO into the gas feed (replacing the 10% N 2 ). This indicates that the large negative current is entirely due to Na 2 SO 3 reduction and that catalytic H + reduction by DdHydAB is completely inhibited under these conditions. Replacing the buffer with fresh Na 2 SO 3 -free buffer decreased the current to the original value observed before addition of Na 2 SO 3 . An analogous experiment at pH 6 ( Figure  7B) showed a small decrease in the current after addition of CO, as well as experiments at pH 7 and pH 8 ( Figure 7C and 7D, respectively), suggesting that at these pH values there is some contribution from the enzymatic H + reduction current, in agreement with the pH dependent formation of SO 2 from For example, at pH 5 (A) the potential was initially set to −109 mV, next stepped down to −459 mV, and finally back to the initial potential −109 mV. Addition of 40 mM Na 2 SO 3 is indicated by red arrows, while addition of 10% CO (in 90% H 2 ) to the gas mixture is indicated by the shaded gray area. After more than 3600 s, the buffer inside the electrochemical cell was rinsed and exchanged with fresh buffer without Na 2 SO 3 . Note the complex behavior in the region immediately following CO treatment at pH 6 in (B). This represents a convolution of the current recovery due to CO release and the exponential decay of the current as a result of decreasing Na 2 SO 3 reduction. To observe the current recovery due to CO release, a simulated exponential decay curve was subtracted from the experimental data ( Figure S17C), and the resulting difference curve is plotted in Figure S17D. Na 2 SO 3 . Control experiments in the complete absence of Na 2 SO 3 showed full inhibition of the electrocatalytic H +reduction current by CO, thus demonstrating that in the absence of Na 2 SO 3 the reductive current is indeed enzymatic H + reduction ( Figure S17). At this stage, it is unclear whether the loss in activity in both directions due to Na 2 SO 3 addition is directly related to the increase in the redox potential of [4Fe-4S] H . The higher redox potential of the cluster may disrupt the proton-coupled electronic rearrangement between [4Fe-4S] H and [2Fe] H . 14 These experiments help to understand the discrepancy between reported H + reduction activity solution assays and electrochemistry. While solution assays (where NaDT is used as electron source) indicate a maximum in activity at pH 7, 8 and almost no activity at pH 5, electrochemical measurements show the highest H + reduction activity at pH 5 ( Figure S15). Regardless, these data show that, under the conditions where H ox H and H red ′H form, the enzyme has lower activity, suggesting that these states are not active intermediates of the catalytic cycle of [FeFe] hydrogenases. This is in stark contrast to the suggestion from Stripp and Haumann that a catalytic cycle involving H ox H is actually the faster branch of the cycle compared to that involving the H red H + and H sred H + states ( Figure 1D).

■ DISCUSSION
In this work we have shown that in CrHydA1 the H ox H state forms in the presence of oxidation products of NaDT at low pH, specifically SO 2 . SO 2 binding caused formation of H ox H not only with CrHydA1 but also with the bacterial enzymes CpHydA1 and DdHydAB, suggesting this is a common behavior in [FeFe] hydrogenases. Additionally, we have shown that with Na 2 SO 3 and in the presence of H 2 the reduced H red ′H state can also form. The electrochemistry measurements showed loss in electrocatalytic activity when DdHydAB was exposed to Na 2 SO 3 , especially at low pH, suggesting that H ox H and H red ′H are less active states and challenging their inclusion in the catalytic cycle. Taken together, these findings suggest that H ox H and H red ′H are not protonated versions of H ox and H red , but instead are forms of H ox and H red in which a product of NaDT oxidation, most likely SO 2 , is bound. Thus, we suggest renaming H ox -DT i and H red -DT I (for dithionite inhibited) to avoid confusion, and for the rest of the discussion we will name them as such.
This result helps explain previous findings in the literature regarding these states. Originally, H ox -DT i and H red -DT i were discovered during NaDT-mediated H + reduction by [FeFe] hydrogenase at low pH. 19,27 Under these conditions H + reduction rates are high, leading to rapid oxidation of NaDT to generate a mixture of SO 3 2− , HSO 3 − , and SO 2 . At low pH, SO 2 forms due to the protonation equilibria and it can bind to the hydrogenase yielding the H ox -DT i and H red -DT i states. It was noticed that the accumulation of H ox -DT i was dependent both on pH and on NaDT concentration, both of which will affect the rate of SO 2 accumulation. Furthermore, it was noted that less active forms of the hydrogenase (e.g., with the PDT cofactor) accumulated H ox -DT i more slowly. In this case, the accumulation of SO 2 depends on the rate of NaDT oxidation by the catalytic activity of the hydrogenase, and it is well established that the PDT-form of the hydrogenase is catalytically much less active than the native ADT-form. 64 Protonation at [4Fe-4S] H is a critical component in the catalytic cycle proposed in Model 2 ( Figure 1D). We recently demonstrated that (in the absence of NaDT) the redox potential of [4Fe-4S] H is pH-independent, challenging the involvement of PCET in the formation of H red and the protonation at [4Fe-4S] H . 12 Figure 1D) is generated by the oxidation products of NaDT. If reduction of [4Fe-4S] H is coupled to protonation then it has to be coupled to protonation in all the steps involving reduction of [4Fe-4S] H . Considering that the hydrogenase enzyme is reversible, with a very low overpotential in either direction, it must be assumed that each step in the catalytic cycle is also reversible and, thus, H ox should be able to protonate to give H ox -DT i . However, incubation of H ox at low pH in the absence of NaDT does not generate H ox -DT i (Figure 2), so H ox -DT i is clearly not a reversibly protonated form of H ox .
Our results also help to explain the misassignment of the pH dependence of the H ox /H red transition. It is important to recall that in this study we also observe that the H ox -DT i /H red -DT i transition is about 60 mV more positive than the H ox /H red transition, as also reported by Senger et al. 10 If the conversion of H ox to H ox -DT i and H red to H red -DT i depend on the pH, then we expect that the "apparent" redox potential of both transitions will shift from the intrinsic redox potential of H ox / H red to the intrinsic redox potential of H ox -DT i /H red -DT i as the pH is decreased. This is simply a consequence of the redox and protonation equilibria being coupled (see Supporting Information and Figure S18 for further details and a model illustrating this behavior). As we demonstrated that the SO 2 concentration in solution increases with decreasing pH and that SO 2 is responsible for binding to H ox /H red to generate H ox -DT i /H red -DT i , then this gives us a pH dependent conversion of H ox /H red to H ox -DT i /H red -DT i and, therefore, an apparent pH dependence of the redox potential.
A further important finding regarding the [FeFe] hydrogenase is the fact that SO 2 appears to inhibit the H 2 oxidation and H + reduction activity of the enzyme. This may be due to the increased redox potential of [4Fe-4S] H . While we do not yet completely understand this effect, it highlights the importance of the balance of redox potentials between the two parts of the H-cluster in facilitating electronic coupling and efficient catalysis. We previously showed that mutation of a cysteine ligating [4Fe-4S] H to histidine increased the redox potential by ∼200 mV. This completely abolished H + reduction activity, while actually enhancing H 2 oxidation at high overpotentials. 65 The In addition to shedding light on the catalytic cycle of [FeFe] hydrogenases, this work reports how NaDT, a compound commonly employed as a reducing agent in metalloenzyme research, is responsible for the generation of artifacts, which were erroneously characterized as catalytically relevant states. To our knowledge, this is the first report of such "noninnocent" behavior of NaDT with [FeFe] hydrogenases, in this case caused by the interaction of one of the NaDT oxidation products with the enzyme. The experimental conditions should, thus, be carefully evaluated when NaDT is chosen as the reducing agent with these enzymes. As we have shown, acidic conditions facilitate formation of H ox -DT i , but at a high concentration of sulfite this state also forms at pH 7. Therefore, particular care must be taken when [FeFe] hydrogenase samples that contain (or contained) NaDT are studied at low pH, or in those cases where NaDT is used as a continuous source of electrons. While this is the first time that NaDT has been shown to interfere with spectroscopic studies of [FeFe] hydrogenases, several previous studies of various other metalloenzymes have reported similar effects. This problematic behavior has been attributed to several factors, from the slow kinetics of NaDT dissociation limiting the catalytic behavior to the unwanted interaction of its oxidation products with the system under study, as we described for [FeFe] hydrogenases. Importantly, the enzymes affected catalyze various reactions and harbor various metallocofactors, suggesting that it is difficult to predict which enzymes will be affected. As such, it is possible that similar effects are still going undetected for other systems. Therefore, the chemistry of NaDT and of its oxidation products should be carefully considered when choosing this compound as a reducing agent for metalloproteins research, and important control experiments should be routinely employed to identify possible sidereactions that can engage with the system under study. In the future it will also be important to evaluate alternative artificial reductants such as Ti(III) citrate (E 0 ′ < −800 mV vs SHE 66 ) and Eu(II)-DPTA (E 0 ′ < −1 V vs SHE 67 ) or the physiological redox partners for their use in hydrogenase research as well as with other metalloproteins.
Inhibition of [FeFe] hydrogenases by sulfite may not simply be an artifact but could represent a physiological mechanism for diverting electrons away from H + reduction by hydrogenase and toward sulfite reduction by dissimilatory and assimilatory sulfite reductases. Here, we showed that the [FeFe] hydrogenases from C. reinhardtii, D. desulf uricans, and C. pasteurianum all form the H ox H state in the presence of sulfite, each of which contains a sulfite reductase. In C. reinhardtii and C. pasteurianum, both [FeFe] hydrogenase and sulfite reductase receive electrons from ferredoxin. 68 ] subcluster and appears to increase the cluster redox potential. This in turn may explain the observed decrease in catalytic activity. Overall, these results highlight the importance of finely tuned redox potentials for catalytic activity and reversibility. More generally, these results should come as a cautionary note to all who use sodium dithionite in metalloprotein studies without concern for its "non-innocent" effects. Sodium dithionite is routinely used in studies on a wide range of metalloenzymes including nitrogenase, CO dehydrogenase, formate dehydrogenase, and many more. Careful evaluation of results from a range of nonphysiological reductants should help to establish the effects that are artifacts from those that represent the physiological behavior of the enzyme of interest.